Molecular programs induced in alveolar resident and recruited mononuclear phagocytes by Toll-like receptor 2/4 agonists

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CabanskiMaciej_2009_01_30.pdf (1.41 MB)

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2009

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http://dx.doi.org/10.22029/jlupub-13450

Abstract

Mononuclear phagocytes play a pivotal role in lung host defence to inhaled pathogens by activation of both innate and adaptive immunity. Resident alveolar macrophages (AMs) are the primary mononuclear phagocytic cells found in the lower respiratory tract that play a central role in regulating pulmonary immune responses. Circulating mononuclear phagocytes (MPs) recruited into inflamed lungs, however are increasingly implicated as essential players in defence against a range of inhaled pathogens. To further elucidate the function of mononuclear phagocytes in regulating pulmonary immune responses we analysed molecular programs induced by bacterial ligands that are recognized by different TLRs. First, we discovered that TLR2 ligand Pam3CSK4 and TLR4 ligand LPS induced in AMs the expression of PKR, previously identified as an essential component of the innate antiviral response. More important we found that both TLR2 and TLR4 agonists induced rapid phosphorylation of PKR strictly dependent on the functionality of the respective TLR. Pharmacologic inhibition of PKR activity using 2-aminopurine (2-AP) and PKR gene deletion were found to reduce the TLR2/4-induced activation of the JNK signalling pathway (MKK4/JNK/c-Jun), but did not affect p38 and ERK1/2 activation. Moreover, inhibition of PKR phosphorylation severely impaired TNF-alpha and IL-6 production by AMs in response to LPS and Pam3CSK4. Additionally, we found that PKR phosphorylation plays a major role in LPS but not Pam3CSK4-induced activation of the p65 subunit of NF-kappaB. Collectively, these results indicate that functional PKR is critically involved in inflammatory responses of primary AMs to gram-positive as well as gram-negative bacteria cell wall components. In addition, we investigated the induction of lung inflammation and the concomitant MP recruitment after alveolar deposition of the TLR2 ligand Pam3CSK4. By using cell sorting, mRNA pre-amplification and whole genome oligonucleotide microarray techniques we found that alveolar trafficking of MPs was associated with profound changes of their gene expression profiles post recruitment (~2500 genes increased). In particular, alveolar recruited MPs showed strong up-regulation for genes encoding cytokines/chemokines, PRR associated molecules and genes involved in eicosanoid metabolism. Interestingly, gene expression profiling revealed that lung recruited MPs displayed simultaneous induction of both pro- and anti-inflammatory genes. However, we observed a dynamic change of the genetic program of MPs found in BALF at different time intervals post challenge. Strong early induction of a subset of pro-inflammatory mediators such as TNF-alpha, CCL2 and IL-6 was found to decrease during the later resolution phase whereas increased transcript levels of central anti-inflammatory and pro-resolution mediators including IL-1RN, IRAK-M, IL-10 and BAX persisted at the same levels. Collectively, our in vivo study identifies for the first time the global genetic program activated in MPs at different time points during TLR2 ligand-induced recruitment to the alveolar space and thus may help to better understand how alveolar recruited MPS may contribute to the development and termination of pneumonia caused by gram-positive bacteria.

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